Research ArticleBasic Science Investigation
Open Access

In Vivo PET Imaging of 89Zr-Labeled Natural Killer Cells and the Modulating Effects of a Therapeutic Antibody

Truc T. Pham, Alicia Chenoweth, Natasha Patel, Arshiya Banu, Gabriel Osborn, Philip J. Blower, Sophia N. Karagiannis and Michelle T. Ma
Journal of Nuclear Medicine June 2024, jnumed.124.267876; DOI: https://doi.org/10.2967/jnumed.124.267876

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  • Figure
  • FIGURE 1.
    FIGURE 1.

    In vitro functional and phenotypic characteristics of 89Zr-NK cells and 89Zr-retention. (A) Flow cytometry shows comparable CD56 and CD16 expression in unlabeled and 89Zr-NK cells. (B) Chemotaxis assays show that chemotactic responses to fetal bovine serum, in unlabeled and 89Zr-NK cells, are similar (mean ± SD, n = 5). (C) Degranulation assays reveal similar degranulation levels in 89Zr-NK and unlabeled NK cells under various conditions (mean ± SD, n = 4). (D and E) Viability (D) and proliferation profiles (E) indicate that both 89Zr-NK and unlabeled NK cells remained viable for up to 7 d in culture without interleukins. Although unlabeled NK cells continued to proliferate, 89Zr-NK cells did not (mean ± SD, n = 7). (F) 89Zr retention in 89Zr-NK cells gradually decreased over 7 d in culture, with 36.4% ± 10.5% of initial activity remaining on day 7 (mean ± SD, n = 7). *P < 0.05. **P < 0.01. ****P < 0.0001. FBS = fetal bovine serum; ns = nonsignificant; PMA = phorbol 12-myristate-13-acetate; RFU = relative fluorescence units.

  • FIGURE 2.
    FIGURE 2.

    ADCC assays using 89Zr-labeled and unlabeled NK cells against HER2-expressing breast cancer cell lines HCC1954 (A) and SKBR3 (B) (10:1 effector/target ratio) and various trastuzumab concentrations. 89Zr-NK cells demonstrated similar ADCC response to unlabeled NK cells. ADCC response varied among human NK cells from different healthy volunteers (Supplemental Fig. 1). Data were fitted to 4-parameter logistic curve.

  • FIGURE 3.
    FIGURE 3.

    Biodistribution of 89Zr-NK cells in female NSG mice bearing orthotopic HER2-expressing HCC1954 tumors. (A) Representative maximum-intensity projection (MIP) and tumor slice PET/CT images of mice administered 89Zr-NK cells (107 cells, ∼150–200 kBq) in combination with trastuzumab (5 mg/kg) or PBS only. Tumors are outlined for clarity. (B) PET image quantification of selected organs and tumors (mean ± SD, n = 4–6/group). *P < 0.05. **P < 0.01. Li = liver; Lu = lungs; MIP = maximum-intensity projection; ns = nonsignificant; Sp = spleen; T = tumor.

  • FIGURE 4.
    FIGURE 4.

    Flow cytometry and γ-counting of CD45-positive and CD45-negative cell populations from murine liver (A) and spleen (B) revealed that 89Zr radioactivity was associated with human CD45-positive (huCD45) cells (n = 4, mean ± SD), which were confirmed as C56-positive/CD16-positive NK cells. CPM = counts per minute; FSC-A = forward scatter area.

  • FIGURE 5.
    FIGURE 5.

    Maximum-intensity projection (MIP) (A) and tumor slice PET/CT and SPECT/CT (B) images of mice 3 d after injection of 89Zr-NK cells in combination with [111In]In-CHX-A″-DTPA-trastuzumab. HU = Hounsfield unit; Sp = spleen; T = tumor.

  • FIGURE 6.
    FIGURE 6.

    Confocal microscopy images (×40 magnification) of lung, spleen, liver, and tumor sections 3 d after injection of NK cells and trastuzumab. Sections stained with DAPI revealed presence of CMFDA–labeled NK cells. Scale bar is 50 μm. CMFDA = 5-chloromethylfluorescein diacetate; DAPI = 4′,6-diamidino-2-phenylindole.

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